Chimeric Norovirus P Particle and Preparation and Use Thereof
Provided is a recombinant P particle formed from a norovirus capsid P protein of a chimeric Aβ1-m peptide (m being an integer ranging from 6 to 15), wherein the recombinant P particles form an ordered and repetitive antigen array. Also provided are a nucleotide sequence encoding the recombinant P particle, a pharmaceutical composition comprising same and a use thereof for preparing a medicament for treating or preventing Alzheimer's disease. Also provided is a method for preparing the recombinant P particle.
The present invention relates to the fields of molecular biology and immunology. In particular, the present invention relates to a recombinant P particle formed from a norovirus capsid P protein of a chimeric Aβ1-m peptide, wherein the recombinant P particles form an ordered and repetitive antigen array, and wherein m is an integer ranging from 6 to 15.
BACKGROUND ARTAlzheimer's disease (AD), also named senile dementia, is a progressive neurodegenerative disease. Its main clinical characteristics include gradual occurrences of memory deterioration, cognitive function disorders and abnormal behaviors, and ending up with a loss of the ability to live independently. AD leads to death from the complications 10-20 years after the onset thereof. Currently, this most common neurodegenerative disease cannot be cured or effectively delayed, and severely jeopardizes the physical and mental health and life quality of the elderly.
Researchers generally believe that the amyloid-β protein (Aβ) in the brain tissue is a primary pathogenic cause of neuronal damage and cognitive function disorders. Aβ is derived from its precursor β-amyloid precursor protein (abbreviated as βAPP). βAPP, as a transmembrane protein, is ubiquitously present in various tissues of the body and expressed most highly in the brain tissue. In the amyloid cleaving pathway, βAPP can be cleaved into Aβ proteins which are 38-43 amino acids in length, wherein Aβ1-42 protein is the main type that forms amyloid deposits.
Aβ active immune vaccine, which is intended to stimulate the body to produce Aβ antibodies and thus clear up Aβ1-42 deposits in the brain, is an important method for treating Alzheimer's disease. As a pioneer of AD vaccines, Aβ1-42 polypeptide vaccine shows a good therapeutic effect of delaying memory decline in the AD transgenic mouse model. Nevertheless, in the clinical trials of Aβ1-42 polypeptide vaccine, 6% of the AD patients showed the side effect of cephalomeningitis, and they had a low level of Aβ1-42 antibody. The research showed that although Aβ1-42 polypeptide vaccine could elicit antibody reactions, it may promote vaccinated patients to produce autoimmune T cell reactions in the brain and thus induce meningoencephalitis as it carries a large number of T cell epitopes. In comparison with Aβ1-42, polypeptide Aβ1-15 comprising only the first 15 amino acids at the N-terminal of Aβ polypeptide, comprises only B cell epitopes instead of T cell epitopes, and this polypeptide vaccine could stimulate the human body to produce specific immunological reactions against Aβ1-42 protein and show good safety in human trials. Therefore, Aβ1-15 has become an antigenic peptide with great research potentials. However, as a hapten, Aβ1-15 does not have a good immunological effect and sustainability of inducing the production of antibodies. Therefore, there are urgent needs for a vaccine against Alzheimer's disease with high safety and good immunological effect.
In the prior art, it is often preferred to synthesize the virus-like particle and Aβ polypeptide respectively, and then couple them in order to obtain vaccines with high immunogenicity. However, as to the vaccines obtained by methods such as coupling, it is difficult to control the number of the inserted epitopes, and to purify the vaccines effectively.
SUMMARY OF THE INVENTIONThe inventors found that the capsid P protein of norovirus is a very ideal antigen exhibition platform for an Aβ1-m (m is an integer ranging from 6 to 15) peptide. When different copies of Aβ1-m (m is an integer ranging from 6 to 15) peptide encoding sequences with various lengths are inserted into the loop of the DNA sequence encoding the capsid P protein of norovirus, Aβ1-m can be exhibited on the utmost surface of the recombinant P particle formed from the recombinant P protein, thereby helping to stimulate the body to the greatest extent to produce specific immunological reactions against Aβ1-42 proteins. The recombinant P particle of the present application can be prepared easily, and are controllable in terms of the number of the inserted epitopes; they can be purified easily and manufactured with a low cost; they have good safety and high immunogenicity, and can induce the production of high-titer antibodies.
In the present invention, multiple copies of DNA encoding antigen peptide Aβ1-m (m is an integer ranging from 6 to 15) are inserted into the DNA encoding loop1, loop2 and/or loop3 of the P protein by means of genetic engineering. By in vitro expression and self-assembly of recombinant proteins, a recombinant P particle capable of sufficiently exhibiting the antigen epitopes of Aβ1-m (m is an integer ranging from 6 to 15) is formed, thereby helping to enhance the efficacy, sustainability and safety of vaccination.
In a first aspect, the present invention provides a recombinant P particle formed from a norovirus capsid P protein of a chimeric Aβ1-m peptide, wherein m is an integer ranging from 6 to 15. In an embodiment of the invention, m may be 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. Said recombinant P particles form an ordered and repetitive antigen array, wherein the amino acid sequence of at least one of the Aβ1-m peptides is embedded into loop1, loop2 and/or loop3 of the norovirus capsid P protein.
In a preferred embodiment of the present invention, the amino acid sequence of a norovirus capsid P protein is shown as SEQ ID NO: 1. In the recombinant P particle, N1 Aβ1-m peptide sequences are embedded behind one or more amino acid sites selected from the group consisting of amino acids 70-74 of SEQ ID NO:1, i.e. I70, A71, G72, T73 and Q74; N2 Aβ1-m peptide sequences are embedded after one or more amino acid sites selected from the group consisting of amino acids 148-151 of SEQ ID NO:1, i.e. T148, S149, N150 and D151; and N3 Aβ1-m peptide sequences are embedded after one or more amino acid sites selected from the group consisting of amino acids 168-171 of SEQ ID NO:1, i.e. D168, G169, S170 and T171; wherein N1, N2 and N3 each are independently selected from an integer ranging from 0 to 40, and N1+N2+N3≥1. In particular, N1, N2 and N3 each are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and N1+N2+N3≥1.
That “Aβ1-m peptide sequences are embedded after one amino acid site” means that the N terminal of the Aβ1-m peptide sequences is directly linked to the C terminal of the amino acid residue via a peptide bond or is linked to the amino acid via an amino acid linker. In a preferred embodiment, the amino acid linker is (Gly)n, wherein preferably n is 1-10, and more preferably n=3.
The multiple consecutive Aβ1-m peptide sequences inserted into SEQ ID NO:1 can be directly linked or linked via an amino acid linker. In a preferred embodiment, the amino acid linker is (Gly)n, wherein preferably n is 1-10, and more preferably n=3.
In the present invention, an Aβ1-m peptide sequence means the polypeptide consisting of the first m amino acids at the N terminal of the entire Aβ1-42 protein, wherein m is an integer ranging from 6 to 15. For example, the Aβ1-m peptide according to the present invention can be a polypeptide consisting of amino acids 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-13, 1-14 or 1-15 at the N terminal of the entire Aβ1-42 protein.
The Aβ1-m peptide sequence according to the present invention can be an Aβ1-m peptide from human, mouse, primate, rabbit, African clawed frog (Xenopuslaevis), rat and guinea pig. The amino acid sequences of Aβ1-15 peptides from human, mouse, primate, rabbit, African clawed frog, rat and guinea pig are shown as SEQ ID NO:2 to SEQ ID NO:8, respectively. According to these sequences, those skilled in the art could easily determine the Aβ1-m peptide (m is an integer ranging from 6 to 15) of the present invention. Preferably, the Aβ1-m peptide is a human Aβ1-m peptide.
In a preferred embodiment of the present invention, the amino acid sequences of the norovirus capsid P proteins of chimeric Aβ1-m peptides are SEQ ID NOs: 15-141. More preferably, the amino acid sequence of the norovirus capsid P protein of a chimeric Aβ1-m peptide is selected from SEQ ID NO:24, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:57, SEQ ID NO:73, SEQ ID NO:92, SEQ ID NO:117, SEQ ID NO:132, SEQ ID NO:133 and SEQ ID NO:134.
In a second aspect, the present invention further provides a nucleotide sequence encoding a norovirus capsid P protein of a chimeric Aβ1-m peptide from which the recombinant P particle of the first aspect is formed, wherein m is an integer ranging from 6 to 15. In an embodiment of the invention, m may be 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. The nucleotide sequence comprises a nucleotide sequence encoding a norovirus capsid P protein and at least one copy of a nucleotide sequence encoding a Aβ1-m peptide, wherein the at least one copy of a nucleotide sequence encoding a Aβ1-m peptide is inserted into the nucleotide sequence encoding a loop of a norovirus capsid P protein, and wherein the inserted nucleotide sequence encoding a Aβ1-m peptide would not result in a frameshift mutation of the norovirus capsid P protein.
In a specific embodiment, the at least one copy of a nucleotide sequence encoding a Aβ1-m peptide can be inserted into the nucleotide sequence encoding the same loop of a norovirus capsid P protein, or into the nucleotide sequences encoding different loops of a norovirus capsid P protein. For example, where the nucleotide sequence encoding a norovirus capsid P protein is SEQ ID NO: 9, loop1 corresponds to nucleotides 208-222, loop2 corresponds to nucleotides 442-453, and loop3 corresponds to nucleotides 502-513.
In a specific embodiment of the invention, the numbers of the Aβ1-m peptide-encoding nucleotide sequences inserted into loop1, loop2 and loop3 are N1, N2 and N3, respectively; N1, N2 and N3 each are independently selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 and 40, and N1+N2+N3≥1.
In a specific embodiment of the invention, the 5′ end nucleotide of the nucleotide sequence encoding a Aβ1-m peptide is linked directly to the nucleotide sequence encoding a loop of a norovirus capsid P protein via 3′,5′-phosphodiester bond or via a DNA linker. The insertion would not result in frame shift during translation of the nucleotide sequence, whether it is a direct insertion or an insertion via a DNA linker.
In a preferred embodiment, the DNA linker is (GGA)n, (GGG)n or (GGC)n, wherein preferably n is 1-10, and more preferably n is 3.
In a specific embodiment of the invention, the multiple consecutive Aβ1-m peptide-encoding nucleotide sequences inserted into the nucleotide sequence encoding a norovirus capsid P protein are linked directly to each other by 3′,5′-phosphodiester bond or by a DNA linker. The insertion would not result in frame shift during translation of the nucleotide sequence, whether it is a direct insertion or an insertion by a DNA linker. In a preferred embodiment, the DNA linker is (GGA)n, (GGG)n or (GGC)n, wherein preferably n is 1-10, and more preferably n is 3.
Preferably, the Aβ1-m peptide nucleotide sequence is a nucleotide sequence encoding an Aβ1-m peptide from human, mouse, primate, rabbit, African clawed frog (Xenopuslaevis), rat and guinea pig. More preferably, the Aβ1-m peptide nucleotide sequence is a nucleotide sequence encoding an Aβ1-m peptide from human.
In a preferred embodiment of the invention, the nucleotide sequences encoding norovirus capsid P proteins of chimeric Aβ1-m peptides are SEQ ID NOs: 142-268. More preferably, the nucleotide sequence encoding a norovirus capsid P protein of a chimeric Aβ1-m peptide is selected from SEQ ID NO: 151, SEQ ID NO: 166, SEQ ID NO: 167, SEQ ID NO: 184, SEQ ID NO: 200, SEQ ID NO: 219, SEQ ID NO: 244, SEQ ID NO: 259, SEQ ID NO: 260 and SEQ ID NO: 261.
In a third aspect, the present invention also provides a pharmaceutical composition used for preventing or treating Alzheimer's disease, comprising the recombinant P particle in the first aspect of the invention and a pharmaceutically acceptable carrier. The pharmaceutical composition is preferably used in mammals, and more preferably used in human. Preferably, the pharmaceutical composition is a vaccine composition, and more preferably, the vaccine composition also comprises an adjuvant. In a specific embodiment of the invention, the adjuvant is CpG. In another specific embodiment of the invention, the adjuvant is an aluminum adjuvant. The vaccine composition is preferably administered subcutaneously or nasally, and more preferably administered subcutaneously.
In a fourth aspect, the present invention also provides use of the recombinant P particle in the first aspect of the invention in the manufacture of a medicament for treating or preventing Alzheimer's disease. Preferably, the medicament is a vaccine. The medicament is preferably used in mammals, and more preferably used in human.
In a fifth aspect, the present invention also provides a method for preparing the recombinant P particle in the first aspect of the invention, comprising the following steps:
i) obtaining an expression vector comprising a nucleic acid encoding a norovirus capsid P protein of a chimeric Aβ1-m peptide, wherein m is an integer ranging from 6 to 15;
ii) transferring the expression vector into a receptor cell;
iii) expressing the norovirus capsid P protein of a chimeric Aβ1-m peptide, and allowing it to self-assemble into a recombinant P particle;
and preferably, the method also comprises isolation and purification steps.
In a specific embodiment, cation exchange chromatography and/or hydrophobic chromatography can be used for purification.
A is the schematic diagram of pET26b-P protein plasmid that has been constructed.
B is the schematic diagram of pET26b-P protein-mEagI plasmid with a mEagI restriction enzyme site obtained by mutation.
C is the schematic diagram of pET26b-P protein-mEagI&mKpnI plasmid with a mEagI restriction enzyme site and a mKpnI restriction enzyme site obtained by mutation.
D is the schematic diagram of pET26b-P protein-10copy-Aβ1-6-loop1G72 plasmid with the gene fragment of interest P protein-10copy-Aβ1-6-loop1G72 loaded between a mKpnI restriction enzyme site and a SalI restriction enzyme site.
E is the schematic diagram of pET26b-P protein-1copy-Aβ1-6-loop2S149 plasmid with the gene fragment of interest P protein-1copy-Aβ1-6-loop2S149 loaded between a mEagI restriction enzyme site and a SalI restriction enzyme site.
F is a schematic diagram of pET26b-Pprotein-10copy-Aβ1-6-loop2S149 plasmid with the gene fragment of interest P protein-10copy-Aβ1-6-loop2S149 loaded between a mEagI restriction enzyme site and a SalI restriction enzyme site.
G is the schematic diagram of pET26b-P protein-20copy-Aβ1-6-loop3G169 plasmid with the gene fragment of interest P protein-20copy-Aβ1-6-loop3G169 loaded between a mEagI restriction enzyme site and a SalI restriction enzyme site.
H is the schematic diagram of pET26b-Pprotein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149 plasmid with the gene fragment of interest Pprotein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149 loaded between a mEagI restriction enzyme site and a mKpnI restriction enzyme site.
I is the schematic diagram of pET26b-P protein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169 plasmid with the gene fragment of interest Pprotein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169 loaded between a mEagI restriction enzyme site and a mKpnI restriction enzyme site.
J is the schematic diagram of pET26b-P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169 plasmid with the gene fragment of interest P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169 loaded between a mEagI restriction enzyme site and a mKpnI restriction enzyme site.
K is the schematic diagram of pET26b-P protein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169 plasmid with the gene fragment of interest Pprotein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169 loaded between a mEagI restriction enzyme site and a mKpnI restriction enzyme site.
L is the schematic diagram of pET26b-P protein-3 copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169 plasmid with the gene fragment of interest P protein-3 copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169 loaded between a mEagI restriction enzyme site and a mKpnI restriction enzyme site.
M is the schematic diagram of pET26b-P protein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169 plasmid with the gene fragment of interest Pprotein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169 loaded between a mEagI restriction enzyme site and a mKpnI restriction enzyme site.
A is the schematic diagram of the locations of three loops of P protein (loop1, loop2 and loop3), together with site-directed mutation sites and restriction enzyme sites carried by P protein gene;
B is the schematic diagram of a P particle gene fragment of interest with 10 copies of Aβ1-6 gene embedded behind site G72 into loop1, abbreviated as P protein-10copy-Aβ1-6-loop1G72;
C is the schematic diagram of a P particle gene fragment of interest with one copy of Aβ1-6 gene embedded behind site S149 into loop2, abbreviated as P protein-1copy-Aβ1-6-loop2S149;
D is the schematic diagram of a P particle gene fragment of interest with 10 copies of Aβ1-6 gene embedded behind site S149 into loop2, abbreviated as P protein-10copy-Aβ1-6-loop2S149;
E is the schematic diagram of a P particle gene fragment of interest with 20 copies of Aβ1-6 gene embedded behind site G169 into loop3, abbreviated as P protein-20copy-Aβ1-6-loop3G169;
F is the schematic diagram of a P particle gene fragment of interest with 10 copies of Aβ1-15 gene embedded behind site G72 into loop1 and 10 copies of Aβ1-6 gene embedded behind site S149 into loop2, abbreviated as P protein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149;
G is the schematic diagram of a P particle gene fragment of interest with 3 copies of Aβ1-12 gene embedded behind site S149 into loop2 and 3 copies of Aβ1-6 gene embedded behind site G169 into loop3, abbreviated as P protein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169;
H is the schematic diagram of a P particle gene fragment of interest with one copy of Aβ1-6 gene embedded behind site G72 into loop1 and 10 copies of Aβ1-6 gene embedded behind site G169 into loop3, abbreviated as P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169;
I is the schematic diagram of a P particle gene fragment of interest with one copy of Aβ1-6 gene embedded behind site G72 into loop1, one copy of Aβ1-6 gene embedded behind site S149 into loop2 and one copy of Aβ1-6 gene embedded behind site G169 into loop3, abbreviated as P protein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169;
J is the schematic diagram of a P particle gene fragment of interest with 3 copies of Aβ1-6 gene embedded behind site G72 into loop1, 3 copies of Aβ1-6 gene embedded behind site S149 into loop2 and 3 copies of Aβ1-6 gene embedded behind site G169 into loop3, abbreviated as P protein-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169; and
K is the schematic diagram of a P particle gene fragment of interest with 10 copies of Aβ1-6 gene embedded behind site G72 into loop1, 10 copies of Aβ1-6 gene embedded behind site S149 into loop2 and 10 copies of Aβ1-6 gene embedded behind site G169 into loop3, abbreviated as P protein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169.
A is the detection of anti-Aβ42 antibody in mouse immune serum after immunization with different dosages of PP-10copy-Aβ1-6-loop2S149 protein vaccine stimulated by different immune adjuvants. B is the detection of T cell responses produced by mouse spleen lymphocytes after immunization with different forms of PP-10copy-Aβ1-6-loop2S149 protein vaccine.
SEQ ID NO: 1 is the amino acid sequence of a norovirus capsid P protein.
SEQ ID NOs: 2-8 are amino acid sequences of Aβ1-15 peptides from human, mouse, primate, rabbit, African clawed frog (Xenopuslaevis), rat and guinea pig.
SEQ ID NO: 9 is the nucleotide sequence encoding a norovirus P protein.
SEQ ID NO: 10 is the nucleotide sequence encoding an A1-15 peptide from human.
SEQ ID NO: 11 is a site-directed mutation forward primer used in the process of obtaining pET26b-P protein-mEagI plasmid.
SEQ ID NO:12 is a site-directed mutation reverse primer used in the process of obtaining pET26b-P protein-mEagI plasmid.
SEQ ID NO: 13 is a site-directed mutation forward primer used in the process of obtaining pET26b-P protein-mEagI&mKpnI plasmid.
SEQ ID NO: 14 is a site-directed mutation reverse primer used in the process of obtaining pET26b-Pprotein-mEagI&mKpnI plasmid.
SEQ ID NO: 15-SEQ ID NO: 141 are nucleotide sequences encoding norovirus capsid P proteins of chimeric Aβ1-m peptides prepared in the examples of the invention.
SEQ ID NO: 142-SEQ ID NO: 151 are nucleotide sequences of DNA fragments of interest synthesized in the process of constructing recombinant plasmids expressing preferred 10 recombinant P proteins, corresponding to 10copy-Aβ1-6-loop1G72, 1copy-AP 1-6-loop2S149, 10copy-Aβ1-6-loop2S149, 20copy-Aβ1-6-loop3G169, 3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169, 10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149, 1copy-AP 1-6-loop1-G72-10copy-Aβ1-6-loop3G169, 1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169, 3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169, and 10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-AP 3-6-loop3G169, respectively.
DETAILED DESCRIPTION OF THE INVENTION DefinitionAll the technical terms used in the present invention have the same meanings as those skilled in the art commonly understand, unless otherwise indicated.
An ordered and repetitive antigen array refers to multiple Aβ1-m peptides (m is an integer ranging from 6 to 15) repetitively and orderly arranged on the surface of a norovirus recombinant P particle.
P protein refers to the P protein in the norovirus capsid P proteins, which can self-assemble in vitro into a P particle. When used herein, P protein used at the gene level means a gene fragment, nucleotide sequence, plasmid, etc. encoding a P protein; P protein used at the protein level means a P protein monomer or dimer. In the accompanying figures, all schematic diagrams of protein show P protein, such as the plasmids encoding P proteins shown in
P particle (abbreviated as PP) refers to a protein particle formed by the in vitro self-assembly of P protein in norovirus. The most common form of P particle is a tetracosamer. When used herein, P particle (PP) only used at the protein level means the form of polymer (such as a tetracosamer), including various proteins used for property detection and immunization, such as the polymer form shown in the denatured electrophoresis of
Pprotein-N1copy-Aβ1-m-loop1Ak1-N2copy-Aβ1-m-loop2Ak2-N3copy-Aβ1-m-loop 3Ak3 means that N1 copies of Aβ1-m are embedded behind the amino acid at position K2 into loop1 of P protein, N2 copies of Aβ1-m are embedded behind the amino acid at position K2 into loop2 of P protein, and N3 copies of Aβ1-m are embedded behind the amino acid at position K3 into loop3 of P protein, wherein m is an integer ranging from 6 to 15. Unless otherwise indicated, Aβ1-m is linked to P protein via a polypeptide linker having three glycines. For example, P protein-1copy-Aβ1-6-loop2T148-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop2N150 means a P protein in which one copy of Aβ1-6 is respectively embedded behind the amino acid at position 148, behind the amino acid at position 149, and behind the amino acid at position 150 into loop2, and each Aβ1-6 is linked to P protein via an amino acid linker having three glycines encoded by GGA.
P protein-10copy-Aβ1-6-loop2S149(GGA)o means that 10 copies of Aβ1-6 are embedded behind the amino acid at position 149 into loop2 of P protein, and there is no linker between Aβ1-6 and P protein, and no amino acid linker between each Aβ1-6, i.e. Aβ1-6 is directly linked to P protein or another Aβ1-6 via a peptide bond.
P protein-10copy-Aβ1-6-loop2S149(GGC)3 means that 10 copies of Aβ1-6 are embedded behind the amino acid at position 149 into loop2 of P protein, Aβ1-6 is linked to P protein via an amino acid linker having three glycines encoded by GGC, and each Aβ1-6 is linked to each other via an amino acid linker having three glycines encoded by GGC.
The technical solutions of the invention are further illustrated by the following examples; however, the present invention is not limited to these examples. Unless otherwise indicated, the formulations and devices used herein are all commercially available.
The present invention provides 127 recombinant P particles in which multiple copies of Aβ1-m peptides (m is an integer ranging from 6 to 15) are embedded into loop1, loop2 and/or loop3 of P protein. The P proteins from which said 127 recombinant P particles are formed are shown in Table 1.
The number of the recombinant P protein or recombinant P particle in the following tables respectively corresponds to the corresponding number of recombinant P protein in Table 1.
The present invention also provides a method for preparing said recombinant P protein particles, comprising the following steps:
1. Synthesizing artificially a DNA fragment comprising a DNA fragment encoding loop1, loop2 and/or loop3 domain(s) of a P protein embedded with multiple copies of Aβ1-m peptide;
2. Constructing pET26b-P protein plasmid (as shown in
3. Carrying out site-directed mutation at the position before loop1 and behind loop3 of P protein by a point mutation method on condition that the amino acid sequence remains unchanged, to obtain the new restriction enzyme sites mKpnI and mEagI (as shown in
4. According to various construction requirements, replacing multiple wild-type circular DNA in its entirety with the synthetic DNA fragment encoding loop1, loop2 and/or loop3 domain(s) of a P protein embedded with multiple copies of Aβ1-m peptide obtained in step 1 by using the restriction enzyme site SalI in the nucleic acid encoding P protein and the obtained enzyme sites mKpnI and mEagI in order to construct various recombinant P protein expression plasmids carrying multiple copies of Aβ1-m peptide respectively (as shown in
5. Transferring the expression plasmids obtained in step 4 into Escherichia coli, where the P protein embedded with an Aβ1-m immunogen is stably expressed and self-assembles into a P particle in the form of a tetracosamer.
Furthermore, according to the present invention, the following analysis and verification experiments were carried out:
1. Ten preferred protein vaccines were characterized by particle diameter determination and electron microscope analysis (as shown in
2. Experiments were carried out with different immune dosages and different immune adjuvants in a C57BL/6J mouse model, using PP-10copy-Aβ1-6-loop2S149 protein vaccine. Results show that when CpG is used as the immune adjuvant, 25 μg protein vaccine could stimulate the mouse to produce the highest titer of a specific antibody against Aβ1-42, and meanwhile would not induce the mouse to produce T cell responses against Aβ1-42 (as shown in
3. Using PP-1copy-Aβ1-6-loop2S149, PP-10copy-Aβ1-6-loop2S149 and PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169 as the immunogens, and CpG as the immune adjuvant, the immunological effects of the three protein vaccines were compared in mouse models, and the effects of both subcutaneous and nasal immunization on the immunological effects of the protein vaccines were analyzed (as shown in
4. Immunization was carried out with 127 candidate proteins. Various proteins were compared for their immunological effects, and the optimal candidate vaccine was screened. Results show that various vaccines can stimulate the mouse to produce a specific antibody against Aβ1-42 compared with the PBS control group; wherein the ten proteins, PP-10copy-Aβ1-6-loopG72, PP-1copy-Aβ1-6-loop2S149, PP-10copy-Aβ1-6-loop2S149, PP-20copy-Aβ1-6-loop3G169, PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149, PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169, PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169, PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169, PP-3 copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169, PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169, have the optimal immunological effects and can stimulate the production of a high concentration of Aβ1-42.
Example 1. Construction of pET26b-P Protein Plasmid1 μL of Nde I enzyme and 1 μL of Xho I enzyme (purchased from Takara Corporation), and 5 μL of enzyme cleavage buffer (purchased from Takara Corporation) were respectively added to 4 μg of pET26b vector plasmid (purchased from Novagen Corporation), and finally sterile water was added to the system to reach a final volume of 50 μL. Digestion was carried out at 37° C. for 5 hours. Then the products were subjected to agarose gel electrophoresis, and recovered using a recovery column (purchased from Invitrogen) to obtain a plasmid vector having double enzyme-digested sticky ends.
A P protein nucleotide sequence as shown in SEQ ID NO: 9 was synthesized by gene synthetic method. The synthetic gene fragment was subjected to Nde I/Xho I double-enzyme digestion using the same method as mentioned above. The products were subjected to agarose gel electrophoresis, and recovered using a recovery column to obtain a gene fragment having double enzyme-digested sticky ends.
The above double enzyme-digested vector fragment and fragment of interest (with a molar ratio of 1:3, and a total volume of 15 μL) were mixed, and 0.75 μL of T4 ligase (purchased from Takara Corporation) and 1.5 μL of ligase buffer (purchased from Takara Corporation) were added. The ligation was carried out at 16° C. overnight to obtain a pET26b-P protein plasmid that can express a P protein particle, as shown in
The site-directed mutation method was as follows:
1. 5′CCGCCG3′ was mutated to 5′CGGCCG3′ by site-directed mutation method using the pET26b-P protein plasmid constructed in Example 1 to obtain pET26b-P protein-mEagI plasmid containing a mEagI restriction enzyme site without altering the amino acid sequence. The specific method was as follows: a pair of perfectly complementary bidirectional primers containing the mutation site was used:
the entire plasmid was subjected to PCR reaction, wherein the PCR reaction system was a KOD-Plus DNA polymerase system (purchased from TOYOBO Corporation), and the total volume of the reaction system was 50 μL (5 μL buffer, 0.2 mM dNTP, 1 mM magnesium sulfate, 0.3 μM upstream primer and 0.3 NM downstream primer, 50 ng template DNA, 1 μL KOD enzyme, and water which was added to a final volume of 50 μL). PCR was carried out in accordance with the reaction system instructions to obtain 20 μL of PCR products. 1 μL of DpnI enzyme (purchased from NEB Corporation) was added to the PCR products. Digestion was carried out at 37° C. for 1 h. 10 μL of the digested products were added to Trant-Blue competent cells (purchased from Peking TransGen Biotech, Ltd.), and placed on ice for 30 min. After that, the cells were subjected to heat shock at 42° C. for 45 s, placed on ice for 2 min. and then were added to 600 μL of fluid LB medium without resistance, and recovered at 200 rpm at 37° C. for 1 h. The culture broth was plated on a LB solid culture plate containing kanamycin (15 μg/mL), and were placed upside down at 37° C. overnight. The obtained mutant plasmid clones were verified by sequencing. pET26b-P protein-mEagI plasmid is shown as
2. 5′GGCACA3′ was mutated to 5′GGTACC3′ using the pET26b-P protein-mEagI recombinant plasmid (as shown in
a pair of perfectly complementary bidirectional primers containing the mutation site was used:
and pET26b-P protein-mEagI&mKpnI plasmid was obtained by the same construction method as mentioned above and verified by sequencing. pET26b-P protein-mEagI&mKpnI plasmid is shown as in
This step involves three different synthesis schemes in total:
The first is a P particle loop gene fragment of interest embedded with a human Aβ1-m gene located between a mKpnI restriction enzyme site and a SalI restriction enzyme site. That is, N1 copies of Aβ1-m gene are merely embedded into loop1. The P protein prepared by this method is abbreviated as P protein-N 1copy-Aβ1-m-loop1.
The second is a P particle loop gene fragment of interest embedded with a human Aβ1-m gene located between a SalI restriction enzyme site and a mEagI restriction enzyme site. That is, (1) N2 copies of Aβ1-m gene are merely embedded into loop2, and the P protein prepared by this method is abbreviated as P protein-N2copy-Aβ1-m-loop2; (2) N3 copies of Aβ1-m gene are merely embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N3copy-Aβ1-m-loop3; and (3) N2 copies of Aβ1-m gene are embedded into loop2 and N3 copies of Aβ1-m gene are embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3, wherein each m is independently selected from an integer ranging from 1 to 40.
The third is a P particle loop gene fragment of interest embedded with a human Aβ1-m gene located between a mKpnI restriction enzyme site and a mEagI restriction enzyme site. That is, (1) N1 copies of Aβ1-m gene are embedded into loop1 and N2 copies of Aβ1-m gene are embedded into loop2, and the P protein prepared by this method is abbreviated as protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2; (2) N1 copies of Aβ1-m gene are embedded into loop1 and N3 copies of Aβ1-m gene are embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N1copy-Aβ1-m-loop1-N3copy-Aβ1-m-loop3; and (3) N1 copies of Aβ1-m gene are embedded into loop1, N2 copies of Aβ1-m gene are embedded into loop2 and N3 copies of Aβ1-m gene are embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2-N3 copy-Aβ1-m-loop3, wherein each m is independently selected from an integer ranging from 1 to 40.
The above three synthesis schemes are illustrated as follows:
3.1 Scheme I:
3.1.1
The loop1 gene fragment embedded with DNA encoding 10 copies of Aβ1-6 peptide of P protein-10copy-Aβ1-6-loop1G72 (as shown in
3.2 Scheme II
3.2.1
The loop2 gene fragment embedded with DNA encoding one copy of Aβ1-6 peptide of P protein-1copy-Aβ1-6-loop2S149 (as shown in
3.2.2
The loop2 gene fragment embedded with DNA encoding 10 copies of Aβ1-6 peptide of P protein-10copy-Aβ1-6-loop2S149 (as shown in
3.2.3
The loop3 gene fragment embedded with DNA encoding 20 copies of Aβ1-6 peptide of P protein-20copy-Aβ1-6-loop3G169 (as shown in
3.2.4
The loop2 gene fragment embedded with DNA encoding 3 copies of Aβ1-12 peptide and loop3 gene fragment embedded with DNA encoding 3 copies of Aβ1-6 peptide of P protein-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169 (as shown in
3.3 Scheme III
3.3.1
The loop1 gene fragment embedded with DNA encoding 10 copies of Aβ1-15 peptide and loop2 gene fragment embedded with DNA encoding 10 copies of Aβ1-6 peptide of P protein-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149 (as shown in
3.3.2
The loop1 gene fragment embedded with DNA encoding 1 copy of Aβ1-6 peptide and loop3 gene fragment embedded with DNA encoding 10 copies of Aβ1-6 peptide of P protein-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3S169 (as shown in
3.3.3
The loop123 gene fragment embedded respectively with DNA encoding 1 copy of Aβ1-6 peptide of P protein-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3G169 (as shown in
3.3.4
The loop, loop2, loop3 gene fragments embedded respectively with DNA encoding 3 copies of Aβ1-6 peptide of P protein-3 copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169 (as shown in
3.3.5
The loop1, loop2, loop3 gene fragments embedded respectively with DNA encoding copies of Aβ1-6 peptide of Pprotein-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169 (as shown in
Furthermore, in addition to the above 10 recombinant P proteins, synthetic methods of other 117 recombinant P proteins respectively involved the above three schemes, as specifically shown in Table 2.
Corresponding to Example 3, this step involves three different synthesis schemes in total:
The first, directed at Scheme I of Example 3, is a P particle loop gene fragment of interest embedded with a human Aβ1-m gene located between a mKpnI restriction enzyme site and a SalI restriction enzyme site. That is, N1 copies of API-m gene are merely embedded into loop1. The P protein prepared by this method is abbreviated as P protein-N1copy-Aβ1-m-loop1.
The second, directed at Scheme II of Example 3, is a P particle loop gene fragment of interest embedded with a human API-m gene located between a SalI restriction enzyme site and a mEagI restriction enzyme site. That is, (1) N2 copies of Aβ1-m gene are merely embedded into loop2, and the P protein prepared by this method is abbreviated as P protein-N2copy-Aβ1-m-loop2; (2) N3 copies of Aβ1-m gene are merely embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N3copy-Aβ1-m-loop3; and (3) N2 copies of Aβ1-m gene are embedded into loop2 and N3 copies of Aβ1-m gene are embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3.
The third, directed at Scheme III of synthesizing a P protein embedded with an API-m immunogen in Example 3, is a P particle loop gene fragment of interest embedded with a human API-m gene located between a mKpnI restriction enzyme site and a mEagI restriction enzyme site. That is, (1) N1 copies of Aβ1-m gene are embedded into loop1 and N2 copies of Aβ1-m gene are embedded into loop2, and the P protein prepared by this method is abbreviated as P protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2; (2) N1 copies of Aβ1-m gene are embedded into loop1 and N3 copies of Aβ1-m gene are embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N1copy-Aβ1-m-loop1-N3copy-Aβ1-m-loop3; and (3) N1 copies of Aβ1-m gene are embedded into loop1, N2 copies of Aβ1-m gene are embedded into loop2 and N3 copies of Aβ1-m gene are embedded into loop3, and the P protein prepared by this method is abbreviated as P protein-N1copy-Aβ1-m-loop1-N2copy-Aβ1-m-loop2-N3copy-Aβ1-m-loop3.
The above three synthesis schemes are illustrated as follows: 4.1 Scheme I: The pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2 was subjected to SalI/KpnI double-enzyme digestion, using the mKpnI restriction enzyme site obtained by mutation and the SalI restriction enzyme site carried by the sequence itself, in order to excise the original loop1 region. The plasmid was recovered as the vector.
Meanwhile, the recombinant loop1 DNA fragment comprising multiple copies of the human Aβ1-m sequence synthesized in Example 3 was subjected to SalI/KpnI double-enzyme digestion in order to obtain the DNA fragment of interest.
The digested DNA fragment of interest was ligated to the digested vector in order to obtain a pET26b plasmid that can express a recombinant P protein embedded with an Aβ1-m immunogen. Finally, the plasmid was verified by sequencing, and thus a correct plasmid was obtained.
4.1.1 Construction of a plasmid vector expressing PP-10copy-Aβ1-6-loop1G72 Protein
1 μL of Sal I enzyme and 1 μL of Kpn I enzyme (purchased from Takara Corporation) and 5 μL of digestion buffer (purchased from Takara Corporation) were added respectively to 4 μg of pET26b-P protein-mEagI-mKpnI vector plasmid obtained in Example 2, and finally sterile water was added to the system to reach a final volume of 50 μL. Digestion was carried out at 37° C. for 5 hours. The digested products were subjected to agarose gel electrophoresis, and recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain a plasmid vector having sticky ends.
Meanwhile, the DNA fragment 10copy-Aβ1-6-loop1G72 synthesized in Example 3 was subjected to SalI/KpnI double-enzyme digestion by the same method to obtain the gene fragment of interest.
The digested vector was mixed with the digested gene fragment of interest. 0.75 μL of T4 ligase (purchased from Takara Corporation) and 1.5 μL of ligase buffer (purchased from Takara Corporation) were added to the mixture. The ligation was carried out at 16° C. overnight to obtain a plasmid vector expressing PP-10copy-Aβ1-6-loop1G72 protein. The plasmid was verified by sequencing, as shown in
4.2 Scheme II
The pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2 was subjected to SalI/EagI double-enzyme digestion, using the mKpnI restriction enzyme site obtained by mutation and the SalI restriction enzyme site carried by the sequence itself, in order to excise the original loop2 and loop3 regions. The plasmid was recovered as the vector.
Meanwhile, the recombinant loop2 and loop3 DNA fragments comprising multiple copies of the human Aβ1-m sequence synthesized in Example 3 was subjected to SalI/EagI double-enzyme digestion to obtain the DNA fragment of interest.
The digested DNA fragment of interest was ligated to the digested vector to obtain a pET26b plasmid that can express a recombinant P protein embedded with an Aβ1-m immunogen. Finally, the plasmid was verified by sequencing, and thus a correct plasmid was obtained.
4.2.1 Construction of a Plasmid Vector Expressing PP-1Copy-Aβ1-6-loop2S149 Protein
1 μL of SalI enzyme and 1 μL of EagI enzyme (purchased from Takara Corporation) and an appropriate amount of digestion buffer (purchased from Takara Corporation) were respectively added to 4 μg of the pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2, and finally sterile water was added to reach a final volume of 50 μL. Digestion was carried out at 37° C. for 5 hours. The digested products were subjected to agarose gel electrophoresis, and recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain a plasmid vector having sticky ends.
Meanwhile, the DNA fragment 1copy-Aβ1-6-loop2S149 synthesized in Example 3 was subjected to SalI/KpnI double-enzyme digestion by the same method to obtain the gene fragment of interest.
The digested vector was mixed with the digested gene fragment of interest. 0.75 μL of T4 ligase (purchased from Takara Corporation) and 1.5 μL of ligase buffer (purchased from Takara Corporation) were added to the mixture. The ligation was carried out at 16° C. overnight to obtain a plasmid vector expressing PP-1copy-Aβ1-6-loop2S149 protein. The plasmid was verified by sequencing, as shown in
4.2.2 Construction of a Plasmid Vector Expressing PP-10Copy-Aβ1-6-loop2S149 Protein
A plasmid vector expressing PP-10copy-Aβ1-6-loop2S149 protein was constructed, using the same method as 4.2.1. The plasmid is shown in
4.2.3 Construction of a Plasmid Vector Expressing PP-20Copy-Aβ1-6-loop3G169 Protein
A plasmid vector expressing PP-20copy-Aβ1-6-loop3G169 protein was constructed, using the same method as 4.2.1. The plasmid is shown in
4.2.4 Construction of a Plasmid Vector Expressing PP-3Copy-Aβ1-12-loopS149-3Copy-Aβ1-6-loop3G169 Protein
A plasmid vector expressing PP-3copy-Aβ1-12-loopS149-3copy-Aβ1-6-loop3G169 protein was constructed, using the same method as 4.2.1. The plasmid is shown in
4.3 Scheme III
The pET26b-P protein-mEagI-mKpnI plasmid vector obtained in Example 2 was subjected to mKpnI/mEagI double-enzyme digestion, using the mEagI restriction enzyme site and the mKpnI restriction enzyme site obtained by mutation, in order to excise the original loop1, loop2 and loop3 regions. The plasmid was recovered as the vector.
Meanwhile, the recombinant loop1, loop2 and loop3 DNA fragments comprising multiple copies of the human Aβ1-m sequence synthesized in Example 3 was subjected to mKpnI/mEagI double-enzyme digestion in order to obtain the DNA fragment of interest.
The digested DNA fragment of interest was ligated to the digested vector in order to obtain a pET26b plasmid that can express a recombinant P protein embedded with an Aβ1-m immunogen. Finally, the plasmid was verified by sequencing, and thus a correct plasmid was obtained.
4.3.1 Construction of a Plasmid Vector Expressing PP-3Copy-Aβ1-6-Loop 1-G72-3Copy-Aβ1-6-loop2S149-3Copy-Aβ1-6-loop3G169 Protein
1 μL of KpnI enzyme and 1 μL of EagI enzyme (purchased from Takara Corporation) and an appropriate amount of digestion buffer (purchased from Takara Corporation) were added respectively to 4 μg of the pET26b-P protein-mEagI-mKpnI vector plasmid obtained in Example 2, and finally sterile water was added to reach a final volume of 50 μL. Digestion was carried out at 37° C. for 5 hours. The digested products were subjected to agarose gel electrophoresis, and recovered using gel recovery kit (purchased from Tiangen Biotech Co., Ltd.) to obtain a plasmid vector having sticky ends.
Meanwhile, the DNA fragment 3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169 synthesized in Example 3 was subjected to EagI/KpnI double-enzyme digestion by the same method to obtain the gene fragment of interest.
The digested vector was mixed with the digested gene fragment of interest. 0.75 μL of T4 ligase (purchased from Takara Corporation) and 1.5 μL of ligase buffer (purchased from Takara Corporation) were added to the mixture. The ligation was carried out at 16° C. overnight to obtain a plasmid vector expressing PP-1copy-Aβ1-6-loop2S149 protein. The plasmid was verified by sequencing, as shown in
4.3.2 Construction of a plasmid vector expressing PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149 protein
A plasmid vector expressing PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149 protein was constructed, using the same method as 4.3.1. The plasmid is shown in
4.3.3 Construction of a Plasmid Vector Expressing PP-1Copy-Aβ1-6-loop1G72-10Copy-Aβ1-6-loop3G169 Protein
A plasmid vector expressing PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169 protein was constructed, using the same method as 4.3.1. The plasmid is shown in
4.3.4 Construction of a Plasmid Vector Expressing PP-1Copy-Aβ1-6-loop1G72-1Copy-Aβ1-6-loop2S149-1Copy-Aβ1l-6-loop3G169 Protein
A plasmid vector expressing PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-11copy-Aβ1-6-loop3G169 protein was constructed, using the same method as 4.3.1. The plasmid is shown in
4.3.5 Construction of a Plasmid Vector Expressing PP-10Copy-Aβ1-6-loop1G72-10Copy-Aβ1-6-loop2S149-10 1Copy-Aβ1-6-loop3G169 Protein
A plasmid vector expressing PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169 protein was constructed, using the same method as 4.3.1. The plasmid is shown in
Furthermore, in addition to the above 10 recombinant P proteins, construction methods of other 117 plasmids expressing recombinant P proteins respectively involve the above three schemes, specifically as shown in Table 2. The obtained recombinant plasmids are not shown.
Example 5 Expression and Purification of a P Protein Embedded with a Human Aβ1-m Immunogen5.1 Expression of a Recombinant P Particle Protein
1 μL of the recombinant plasmids prepared in the above examples were respectively added to 100 μL of E. coli BL21 competent cells (purchased from TransGen Corporation), ice-bathed for 30 min, heat shocked for 90 s in a water-bath at 42° C., and then ice-bathed for 2 min. 600 μL of LB medium was added to the mixture, and cultured at 180 rpm/min at 37° C. for 1 h. The mixture was coated evenly on a LB solid medium containing kanamycin (15 μg/mL) resistance and cultured at 37° C. for 24 h to obtain strains that can stably express recombinant proteins. A growing colony was picked and inoculated into 20 mL of LB medium. The mixture was cultured at 220 rpm at 37° C. When the OD value of the culture mixture reached 1.0, induction by Isopropyl β-D-Thiogalactoside (IPTG at a final concentration of 0.33 mmol/L) was carried out at 220 rpm at 16° C. overnight. After the induction, the culture broth was centrifuged at 4000 rpm for 20 min. The supernatant was discarded, and the bacterial precipitates were resuspended with PBS. Centrifugation was conducted again at 4000 rpm for 20 min and the supernatant was discarded to obtain the bacterial precipitates containing proteins of interest.
5.2 Extraction and Purification of Recombinant P Particle Proteins
The bacterial precipitates obtained in 5.1 were resuspended by adding 20 mL of protein buffer (pH8.0, containing 50 mM Tris and 300 mM KCl). The bacteria were lysed by sonication on ice for 30 min. The mixture was centrifuged at 12,000 rpm at 4° C. for 30 min. Subsequently, the supernatant was taken and allowed to pass through 0.45 μm filter membrane to obtain crude extracts of proteins.
The structures of recombinant P particle proteins are shown as
The crude extracts of proteins were purified using a cation exchange column (purchased from GE Corporation). The specific scheme was as follows: First, the exchange column was rinsed with ultrapure water in a volume of about 100 mL, followed by equilibration with PB solution (pH 5.0) at a flow rate of 2 mL/min. Then 20 mL of the crude extracts of proteins were added to the exchange column at a flow rate of 1 ml/min. After the sample was completely loaded onto the column, the exchange column was rinsed with PB solution (pH 7.0) to remove protein impurities, and then eluted with PB solution containing 1 mol/L NaCl. The proteins at peak value were collected to obtain the proteins of interest.
The proteins were further purified by a hydrophobic chromatography column (purchased from GE Corporation). The specific scheme was as follows: First, the column was rinsed with ultrapure water, and then with PB solution (pH 7.0) at a flow rate of 2 mL/min. After the column was equilibrated well, the protein sample was loaded onto it. After the sample was completely loaded onto the column, the column was eluted by gradient with PB (pH 7.0) and 1 mol/L NaCl solution for 2 hours. The concentration of NaCl decreases from 1 mol/L to 0.1 mol/L. The proteins were collected at peak value.
The sizes of 10 P particle protein monomers were identified by reductive SDS-PAGE. The upper panels of
Then the tetracosamers of P protein particles were further isolated and purified using a Superdex 200 molecular sieve (purchased from GE Corporation). The procedures were as follows: The column was rinsed with ultrapure water at a flow rate of 1 mL/min for one volume of the column, and then again with about 120 mL of PB buffer (pH 5). After that, 2 mL of protein extraction solution was added to the column and washed with PB buffer at a flow rate of 1 mL/min. The proteins at peak value were collected to obtain the tetracosamers of P particle proteins. Three proteins were tested for their polymer structures by native polyacrylamide gel electrophoresis, as shown in the middle panels of
Inventors further analyzed the particle diameter and morphology of 10 protein polymers. The particle diameter was tested using a nanoparticle diameter analyzer (purchased from Malvern Corporation) in accordance with the instructions of the manufacturer. The analysis results showed that there were particles having an average diameter of about 20 nm in the solution of the above 10 recombinant P particles. As shown in the lower panels of
The sizes and P particle diameters of the 127 P particle proteins according to the invention are shown as Table 3.
3. Immunological Effects of Recombinant P Particle Protein Vaccines
3.1 Determination of Immune Dosages and Adjuvants of P Particle Protein Vaccines
The PP-10copy-Aβ1-6-loop2S149 protein vaccine was selected for use in female C57BL/6 mice aged 6-8 weeks (purchased from Beijing HFK Bioscience Co., LTD) to determine the immune dosage and immune adjuvant. The immune dosages were respectively 12.5 μg, 25 μg and 50 μg/animal, and were increased to 100 μL/animal with sterile PBS. Each group contained 6 mice. The immunization was performed by subcutaneous injection. The immune adjuvants were an aluminium adjuvant (purchased from Brenntag Biosector Corporation) and an CpG adjuvant that can specifically stimulate the body to produce humoral immunity (purchased from Takara Corporation). The nucleotide sequence of CpG adjuvant was as follows: TGTCGTCGTCGTTTGTCGTTTGTCGTT. The immunization was carried out at day 1, day 15 and day 29 (three times in total). Blood was taken on the day before every immunization. The mice were sacrificed two weeks after the third immunization. The recombinant P particle protein vaccine was tested for immunological responses of the induced humoral immunity and cellular immunity in mice. There were 7 test groups and 3 control groups. The immune dosage, adjuvant and immunizing antigen for each group are shown in Table 4.
3.1.1 Humoral Immunity-ELISA Detection Experiment
Tails of the mice were cut and blood was taken on the day before every immunization. Blood samples were placed at 37° C. for 2 h, then placed at 4° C. for 1 h, and centrifuged at 3,000 rpm to take the supernatant serum. The serum was frozen for use. Aβ1-42 (purchased from GL Biochem (Shanghai) Ltd.) was used as antigen and formulated into 1 mg/mL solution with sterile PBS. The solution was diluted to 1 ng/L with antigen coating solution and used for coating a 96-well plate (100 μL/well). The plate was coated at 4° C. overnight. After each well was washed three times with 300 μL of PBST (pH 7.4, 0.01 mol/L PBS, containing 0.05% Tween-20), blocking solution (pH 7.4, 0.01 mol/L PBS, 20% fetal bovine serum) was added. Blocking was carried out at 37° C. for 2 hours. Each well was washed three times with PBST. To the wells were added different dilution gradients (1:200, 1:800, 1:3200, 1:12800, 1:51200 and 1:204800) of antiserum (100 μL/well), and incubation was carried out at 37° C. for 1 hour. Each well was washed three times with PBST. To the wells were added 0.3 μg/ml of HPR (horse radish peroxidase) goat-anti-mouse secondary antibody (purchased from Beijing Dingguochangsheng Biotechnology Co. LTD) (100 μL/well), and incubation was carried out at 37° C. for 1 hour. Each well was washed three times with PBST. To the wells were added the substrate tetramethylbenzidine (TMB) (purchased from Tiangen Biotech Co., Ltd.) (100 μL/well), and color was developed in the dark for 25 min. 50 μL of 2M sulfuric acid was added to each well to terminate the reaction. Absorbance was detected at 450 nm using a microplate reader (purchased from Bio-red Corporation).
Experimental results are shown in
3.1.2 Cellular Immunity-ELISPOT Detection
A 96-well plate was coated with the monoclonal antibody against cytokine interferon γ (from elispot kit purchased from BD Corporation) in a concentration of 5 μg/mL (50 μL/well), and covered at 4° C. overnight. After discarding the coating antibody and washing once with a complete medium containing 10% fetal bovine serum, 200 μL of this complete medium was added to each well. Blocking was carried out at 37° C. for 1 hour, and then the medium was discarded. Mice used in the experiment were sacrificed by neck-pulling. Spleen cells of the mice were taken out and formulated into a cell suspension in a concentration of 107/mL. The cell suspension was added to the coated 96-well plate (100 μL/well). 100 μL of 1 μg/mL specific antigen Aβ1-42 was added to each well. The plate was then cultured at 37° C. in an incubator containing 5% CO2 for 24 h to stimulate and activate the cells. 24 h later, the plate was washed two times with sterile water, and six times with sterile PBST (pH7.4, 0.01 mol/L PBS, containing 0.05% Tween-20) buffer to wash the cells away. 50 μL of 2 μg/mL antibody against interferon γ (from elispot kit purchased from BD Corporation) was added to each well and incubated at room temperature for 2 hours. The 96-well plate was washed, and horse radish peroxidase labeled biotin secondary antibody (from elispot kit purchased from BD Corporation) was added (50 μL/well). The plate was cultured at room temperature for 2 h, and washed four times with PBST, and two times with PBS. 50 μL of Elispot color developing solution (AEC substrate) was added to each well and reacted in the dark at room temperature for 5-60 min. The staining solution was discarded, and the plate was washed with distilled water. After being dried overnight, the sample was calculated for the number of activated cells using a microscope.
The results are shown in
3.2 Comparison of Immunological Effects of Three Recombinant P Particle Protein Vaccines
After the experiments for determining immune dosages and immune adjuvants of protein vaccines, the applicant first selected three representative recombinant proteins, adopted the strategy of a protein vaccine dosage of 25 μg/animal and CpG as the adjuvant, compared immunological effects of the three recombinant P particle protein vaccines in female C57BL/6 mice aged 6-8 weeks by the two immunization routes via nasal and subcutaneous injections in order to compare the effects of nasal and subcutaneous immunizations, and identify the differences in immunological effects of various proteins. Similar to the method in 3.1.1, immunization was carried out every two weeks and four times in total. Tails of the mice were cut and blood was taken before every immunization. Serum was tested by ELISA detection. The mice were divided into 7 test groups and 3 control groups. The immunogen and immunization route for each group are shown as in
An ELISA plate was coated with Aβ1-42 as the antigen, using the method described in 3.1.1. A reaction was carried out using the immunized mouse serum as the primary antibody, HRP goat-anti-mouse antibody as the secondary antibody, TMB as the substrate, and sulfuric acid as the termination solution. After the termination of reaction, absorbance was detected at 450 nm using a microplate reader. The contents of antibodies in the serum were compared based on the absorbance. Experiment was carried out using PBS as the negative control, and the commercial antibody 6e10 (purchased from Covance Corporation) as the positive control, and using the sera before and after immunizing mice with three proteins as the test groups. Results are shown in
3.3 Comparison of Immunological Effects of Various Recombinant P Particle Protein Vaccines
The applicant determined that the adopted immune dosage was 25 μg/animal and the immunization route was subcutaneous injection by the experiments for determining immune dosages and immune adjuvants of protein vaccines, and immunological experiments of three representative recombinant proteins, and determined the immunological effects of 127 candidate vaccines in female C57BL/6 mice aged 6-8 weeks. Immunization procedures and method are as described in 3.2. Comparison results of immunological effects are shown in Table 6.
In the above P particles, the 17 P particles, PP-10copy-Aβ1-6-loop1G72, PP-1copy-Aβ1-6-loop2S149, PP-10copy-Aβ1-6-loop2S149, PP-10copy-Aβ1-6-loop3G169, PP-20copy-Aβ1-6-loop3G169, PP-3copy-Aβ1-6-loop1G72-3 copy-Aβ1-6-loop2S149, PP-10copy-Aβ1-15-loop1G72-10copy-Aβ1-6-loop2S149, PP-20copy-Aβ1-15-loop1G72-40copy-Aβ1-6-loop2S149, PP-3copy-Aβ1-12-loop2S149-3copy-Aβ1-6-loop3G169, PP-1 copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169, PP-20copy-Aβ1-12-loop2S149-10copy-Aβ1-6-loop3G169, PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-12-loop3G169, PP-1copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop3G169, PP-3copy-Aβ1-6-loop1G72-10copy-Aβ1-12-loop3G169, PP-1copy-Aβ1-6-loop1G72-1copy-Aβ1-6-loop2S149-1copy-Aβ1-6-loop3 G169, PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169, PP-10copy-Aβ1-6-loop1G72-10copy-Aβ1-6-loop2S149-10copy-Aβ1-6-loop3G169, have good immunological effects and can induce the production of high concentrations of antibodies.
The standard curve is plotted in accordance with positive antibody 6e10, and its linear fitting curve is y=47.692x+0.2964, R2=0.99. According to this standard curve, the concentration of the antibody produced by induction can be calculated within the linear range, wherein PP-3copy-Aβ1-6-loop1G72-3copy-Aβ1-6-loop2S149-3copy-Aβ1-6-loop3G169 produce antibodies in a concentration of about 245.12 μg/mL after the fourth immunization. Therefore, the present invention has good immunological effects and induces a high concentration of Aβ1-42 antibody in mouse serum after immunization. The present invention has very good therapeutical effects and is a protein vaccine with great potentials for treating AD.
Claims
1. A recombinant P particle formed from a norovirus capsid P protein of a chimeric Aβ1-m peptide, wherein m is an integer ranging from 6 to 15, the recombinant P particle forms an ordered and repetitive antigen array, the amino acid sequence of at least one of the Aβ1-m peptide is embedded into loop1, loop2 and/or loop3 of the norovirus capsid P protein, and preferably, the amino acid sequence of the norovirus capsid P protein is shown in SEQ ID NO: 1.
2. The recombinant P particle according to claim 1, wherein N1 Aβ1-m peptide sequences are embedded behind one or more amino acid sites selected from the group consisting of amino acids 70-74 of SEQ ID NO:1, i.e. I70, A71, G72, T73 and Q74; N2 Aβ1-m peptide sequences are embedded behind one or more amino acid sites selected from the group consisting of amino acids 148-151 of SEQ ID NO:1, i.e. T148, S149, N150 and D151; and N3 Aβ1-m peptide sequences are embedded behind one or more amino acid sites selected from the group consisting of amino acids 168-171 of SEQ ID NO:1, i.e. D168, G169, S170 and T171; wherein N1, N2 and N3 each are independently selected from an integer ranging from 0-40, and N1+N2+N3≥1.
3. The recombinant P particle according to claim 1, wherein multiple consecutive Aβ1-m peptide sequences embedded into the norovirus capsid P protein are linked directly or via a polypeptide linker, wherein the polypeptide linker is preferably (Gly)n, preferably n being 1-10, and more preferably n being 3.
4. The recombinant P particle according to claim 1, wherein the Aβ1-m peptide is linked to the norovirus capsid P protein directly or via a polypeptide linker, wherein the 2 of 6 polypeptide linker is preferably (Gly)n, preferably n being 1-10, and more preferably n being 3.
5. The recombinant P particle according to claim 1, wherein the Aβ1-m peptide sequence is an amino acid sequence comprised in sequences selected from SEQ ID NOs: 2-8, and preferably, the Aβ1-m peptide sequence is comprised in SEQ ID NO: 2.
6. The recombinant P particle according to claim 1, wherein the norovirus capsid P protein of a chimeric Aβ1-m peptide is encoded by nucleic acid sequences of SEQ ID NO: 15-SEQ ID NO: 141; preferably, the norovirus capsid P protein of a chimeric Aβ1-m peptide is encoded by a nucleic sequence of SEQ ID NO:24, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:57, SEQ ID NO:73, SEQ ID NO:92, SEQ ID NO:117, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:142.
7. A nucleic acid encoding the recombinant P particle according to claim 1, wherein preferably the nucleic acid has sequences of SEQ ID NO: 15-SEQ ID NO: 141, and more preferably, the nucleic acid has a sequence of SEQ ID NO:24, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:57, SEQ ID NO:73, SEQ ID NO:92, SEQ ID NO:117, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, or SEQ ID NO:142.
8. A pharmaceutical composition used for preventing or treating Alzheimer's disease, comprising the recombinant P particle according claim 1 and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is preferably used in mammals, and more preferably used in human; preferably, the pharmaceutical composition is a vaccine, more preferably, the vaccine also comprises an adjuvant, and most preferably, the vaccine also comprises a CpG adjuvant or aluminum adjuvant; the vaccine is preferably administered for immunization subcutaneously or nasally, and more preferably administered subcutaneously.
9. Use of the recombinant P particle according to claim 1 in the manufacture of a medicament for treating or preventing Alzheimer's disease, wherein preferably the medicament is a vaccine.
10. A method for preparing the recombinant P particle according to claim 1, comprising the following steps:
- i) obtaining an expression vector comprising a nucleic acid encoding a norovirus capsid P protein of a chimeric Aβ1-m peptide, wherein m is an integer ranging from 6 to 15;
- ii) transferring the expression vector into a receptor cell;
- iii) expressing the norovirus capsid P protein of a chimeric Aβ1-m peptide, and allowing it to self-assemble into a recombinant P particle,
- and preferably, the method also comprises isolation and purification steps, and more preferably, the purification step includes using cation exchange chromatography and/or hydrophobic chromatography for purification.
Type: Application
Filed: Jun 29, 2016
Publication Date: Aug 15, 2019
Patent Grant number: 11116831
Inventors: Wei KONG (Changchun), Hui WU (Changchun), Chunlai JIANG (Changchun), Xianghui YU (Changchun), Lu FU (Changchun), Yingnan LI (Changchun)
Application Number: 15/744,585
